In hyaline cartilage type IX collagen there is a minor constituent of the fiber network having type II collagen as a major constituent. The type IX collagen molecule is a heterotrimer consisting of polypeptide chains α1, α2, and α3 (2). It belongs to the fibril-associated collagens with interrupted triple-helix (FACIT). Each chain contain three triple-helical (collagenous) domains COL 1, 2, and 3 surrounded by four non-triple-helical domains, NC 1, 2, 3, and 4 (3), (see also FIG. 9). The domain numbers are counted from the C-terminal.
Using electron microscopy, it has been shown that type IX collagen decorates the surface of type II collagen fibrils and that the NC 4 domain forms a globular structure, which together with the stalk like COL 3 domain protrudes out from the type II collagen fibril (4).
Type IX collagen is covalently crosslinked to the type II collagen fibrils through binding to both type II collagen and other type IX collagen molecules (5-7). These bonds render extraction of type IX collagen from mature cartilage virtually impossible by agents that do not cleave peptide bonds.
The NC 4 domain has been shown to have an affinity for a number of molecules, for example heparin and cartilage oligomeric matrix protein (COMP) (8-10), whereas COL domains interacts with matrilin-3 (11). Mutations in the interactive COMP, MATN-3, and COL9 have been found in patients with multiple epiphyseal dysplasia, a rare inherited heterogeneous range of diseases, affecting growth and ossification of the epiphysis (12).
NC4-fragments have now surprisingly been shown to be biomarkers for cartilage destruction. The cleavage occurs after aggrecan, a major structural component of cartilage is released, and prior to the major collagen degradation. NC4-fragments are markers of the process leading to destruction before the “point of no return” in contrast to collagen-markers that are like x-rays observations a result of the late stages of the destruction process. Aggrecan-related markers are released in an early stage of the degradation progress and also during normal adaptation of the tissue and therefore do not specifically represent a pathological process. Furthermore aggrecan markers are often associated to inflammation.
After the cleavage of NC4, described in this invention, the large part of type IX collagen that remains crosslinked to type II collagen is potentially further cleaved and released at a later stage. The neoepitope antibody disclosed in the invention herein, against the new C-terminal of the released fragment could detect such cleavage. Additionally, neoepitope antiserum disclosed in the invention, could be used in immunohistochemistry to determine were in the cartilage (superficial-deep, pericellular-interterritorial) the novel cleavage reported here has occurred. An antibody to the newly formed N-terminal of the fragment initially remaining in the tissue could detect a later stage release of such fragments. Additional information on timing could be obtained using the neoepitopes created by cleavage in the col3 domain. A number of earlier publications and patent-applications describe monoclonal antibodies that bind specifically to Type IX collagen, but none of them mentions neoepitope antibodies specifically against cleavage sites near NC4.
Patent application WO1990008195 A1 discloses oligopeptides corresponding to segments of human Type IX collagen useful as antigens in producing monoclonal antibodies which bind immunologically to human Type IX collagen and fragments derived from it. However the segments mentioned in the patent application are not involved in pathological destruction of collagen IX, and therefore not expected in connection to arthritis.
In WO2004110475 A1, assigned, Inst of Nutraceutical Research PTY, the invention relates to compositions comprising the NC4 domain, for treatment and tolerance induction to individuals with symptoms of arthritis. The inventors are also describing methods for recovering polypeptides from connective tissue, having anti-arthritic or anti-inflammatory activity. As in the above-mentioned patent-application the antibody is against uncleaved Collagen IX and not a neoepitope antibody. Furthermore the applicants do not mention NC4 as a marker for diagnosis.
The patent-application WO1999021011 A1, assigned Fibrogen Inc, reveals a method for detecting or monitoring autoimmune disorders and connective tissue disorders by determining the levels of type IX collagen in serum. When preparing antibodies against type IX collagen a sequence in the N-terminal portion of the NC4 domain was selected. This antibody is against uncleaved Collagen IX and not to unique cleavage neoepitopes like in the invention herein.
None of the previous documents describes a specific cleavage site occurring in the NC-4 domain leading to exclusive release of this domain. Although the fact that tissues are completely destroyed in certain diseases, the character of the cleavage cannot be inferred or predicted. It was also unexpected that the cleavage of the molecule in bovine cartilage and human cartilage is different, particularly in view of the sequence similarities (FIG. 10).
Thus, the discovery of specific cleavage sites is novel and opens up for the new diagnostic inventions.
Therefore we have developed a sensitive Western blot technique and a sensitive inhibition ELISA as methods for the identified novel cleavage site facilitating analysis of collagen IX destruction in any human sample, such as synovial fluid, serum, urine and any body fluids. This kind of assay could be used to establish if and when the identified cleavage appear in cartilage degradation and joint deterioration in patients. By using the specific neoepitope antibody of the invention herein, the assay can be used for diagnosis of disease progression and monitoring disease response of treatment. For example to determine the optimal moment for a sportsman with a healing bone fracture to return to the training program.